The nerve growth factor I-B (NGFI-B) family of orphan receptors include NGFI-B? (Nur77, TR3), NGFI-B? (Nurr1), and NGFI-B? (Nor1), and ligands for NGFI-B proteins have not been reported. Apoptosis/differentiation inducing agents activate Nur77-dependent apoptosis via nuclear (transactivation) pathways or by nuclear-mitochondrial or nuclear-cytosolic translocation of Nur77. We have now identified for the first time at least three compounds that activate wild-type Nur77 in transactivation assays, and this response requires the ligand binding domain (LBD) of Nur77. The Nur77-active 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes contain CF3 (DIM-C-pPhCF3), H (DIM-C-Ph), and OCH3 (DIM-C-pPhOCH3) substituents. These C-substituted diindolylmethanes (DIMs) induce apoptosis in pancreatic cancer cells through a nuclear pathway, and we hypothesize that C-substituted DIMs represent a unique class of ligands that activate Nur77 and induce apoptosis in pancreatic cancer cells. In Aim 1, the structure-dependent activation of Nur77 by a series of ring- and phenyl-substituted derivatives of C-substituted DIMs will be determined in six pancreatic cancer cell lines that express Nur77 protein. The most active compounds (at least 3) will be used as prototypical Nur77 agonists for investigating ligand-dependent Nur77-DNA binding in electrophoretic mobility shift assay and ligand-induced Nur77-coactivator interactions and enhanced transactivation. Aim 2 will focus on induction of apoptotic pathways by Nur77 agonists and compare these responses to those observed for apoptotic inducing agents (TPA, butyrate and the retinoid CD437) in selected pancreatic cancer cell lines. This approach will resolve apparent differences in previous reports and distinguish between ligand-activated Nur77 (nuclear) vs. extranuclear pathways for induction of apoptosis. Moreover, the molecular mechanisms of Nur77 agonist-induced expression of pro-apoptotic genes will also be investigated. In Aim 3, the in vivo antitumorigenic activity of Nur77 agonists will be determined. The proposed studies will define the mechanism of action of Nur77 activation by selected C-substituted DIMs that are the first known ligands for this orphan receptor. These studies will serve to highlight the potential role of selective Nur77 modulators as a new class of chemotherapeutic drugs for treatment of pancreatic cancer.